Isolation And Identification Of Probiotics From Gut Of Flounder And Its Use In Aquaculture

Three strains of probiotics isolated from the autochthonous flora in gastrointestinal tract of healthy flounders, the adhesion of L.rhamnosus P15 to the skin and enteron mucus of Flounder by H3-TdR labeled and FITC labeled methods, and the effect of L.rhamnosus P15, Bacillus Y2-2 and Y1-13 in culture of flouder were investigated.In experiment 1,experiment2 and experiment 3,by common medium and selective medium, we screened 6 strains of Lactic acid bacteria, 6 strains of antagonistic bacteria against Vibrio sp., and 24 strains of proteinase-producing bacteria from the autochthonous flora in gastrointestinal tract of healthy flounders. Among 6 strains of Lactic acid bacteria, strain P15 was identified to be L.rhamnosus by physiological, biochemical and molecular biological of 16s rRNA methods. L.rhamnosus P15 had strong inhibition effect on Vibrio, and showed good growth potential and powerful inhibition activity in pH6.8 and pH7.5, while no any inhibition activity was detected in pH8.0 and pH8.5. Among 6 strains of antagonistic bacteria against Vibrio sp. screened by dot-inoculation assays from gastrointestinal tract of flounders, no strains except Yl-13 showed antibacterial activities in supernatant inhibition assay. In coculture assay, the number of Vibrio in tests was lower than the control throughout the experiments. It was demonstrated that the Yl-13 was powerful antagonistic against Vibrio. 24 strains of proteinase-producing bacteria were screened by casein enzyme-produced assay and skimmed milk enzyme-produced assay. Proteinase activity was determined by Folin-phenol method. It was indicated that the 24 strains have proteinase activity, in particularly strain E14, F1, G7 and Y2-2.In experiment 4 , experiment 5 , we investigated the adhesion of two strains of L.rhamnosus P15 and L. Casei, the inhibitory adhesion to the skin and enteron mucus of Flounder by H3-TdR labeling and FITC labeling methods .Meanwhile, the changes of pH in the process of strains culture were detected. The growth and change of pH of two lactobacillus strains of the results indicated that Lactobacillus rhammosus was in the exponential phase in 25h-75h incubation, then in the platform phase; while the exponential phase of Lactobacillus casei occurred before 48h of incubation, then in the platform phase. The pH decrease occurred during the culture of L.rhamnosus P15 and L. Casei, and the pH decrease more sharply in culture of L. Casei. Results of Adhesion showed that either L.rhamnosus P15 or L. Casei could adhere to the mucus of skin and enteron, and greater adhesion to stomach mucus of flounder than to the other mucus was found in both strains by H3-TdR and FITC labeling methods (48.18% and 63.0% in L.rhamnosus P15 and L. Casei using FITC labeling methods,respectively), and smaller adhesion rate of both strains was found in mucus of skin using two methods. Using FITC labeling methods, adhesion rate of L.rhamnosus P15 in the mucus of intestine was 19.04% compared with 57.72% of L. Casei. L. Casei showed better adhesion to the mucus than. L.rhamnosus P15 using two methods. L.rhamnosus P15 showed greater adhesion inhibition on Candida albicans and P.Fluorescens in mucus of caecum, decreased adhesion rate by 19.01% compared with normal adhesion of P.Fluorescens, however V.cholerae non-01 strains, Aeromonas hydrophila, decreased by 5.22%,3.22%, respectively. L. Casei had greater inhibition to adhesion of V.cholerae non-01, and decreased adhesion rate by 22.31% compared with normal adhesion of V.cholerae non-01, decreased adhesion rate of P.Fluorescens, Aeromonas hydrophila, Candida albicans by 17.50%> 12.69% and 5.15% ,respectively. The four pathogenic bacteria adhered to the enteron mucus better than to the skin mucus.In experiment 6,A feeding trial using 90 flounder was conducted to study microbiota of fish and colonization of two lactobacillus after feeding the flounder diet with two lactobacillus strains in 1.2 109 cfu/g for 20 days .CFU of two lactobacillus strains reached 106 cfu/g from 103cfu/g in the gastrointestine after 5 day, thenc...